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Characterization of gene induction and antiviral effects on HCVcc following ribavirin, interferon and polyIC stimulation
Abstracts / Cytokine 48 (2009) 46–90 Although the response of cells to type I interferons (IFN) has been extensively studied in vitro, limited information is available on the spatial and temporal activation pattern in vivo. To determine the kinetics and to quantify the response of different cell types to type I IFN, we created BAC transgenic mice expressing firefly luciferase that is transcriptionally controlled by the Mx2 gene promoter. Expression of the reporter with regard to strength and onset kinetics of expression parallels that of Mx2 in cells from transgenic mice. By using in vivo bioluminescence imaging and quantitative reporter gene detection of isolated cells and organs we determined the temporal and spatial propagation of IFN action during viral infection and after administration of ubiquitous IFN inducers. Several unexpected features of the IFN response were observed: Independent of their site of induction, the IFN response is found in blood cells and few organs with the liver as the main target, indicating that blood circulation is the distributor of this cytokine. IFN subtypes a and b and k show clear differences with respect to their effects on organs and kinetics of maximal activity. The results further confirm the constitutive production of IFN in adult animals and show that also this response is found predominantly in the liver. Infection of reporter mice with different strains of influenza virus uncovers two main features of the IFN response: (i) Type I IFNs wherever they are produced are rapidly brought into circulation; and (ii) Infection of mice with a pathogenic virus strain does not necessarily lead to a complete block of IFN response but rather change the spatial and temporal pattern of IFN activity. In reporter mice crossed to IFNAR1 deficient mice the targets of IFN-k could be identified indicating that type III IFN acts mainly on mucosal surfaces. doi:10.1016/j.cyto.2009.07.303
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the induction of interferon-stimulated genes (ISGs) by interferon in vitro and in vivo. Global gene expression profiles generated from the Huh7.5.1 cell line, treated with or without ribavirin, were analyzed using GeneGO and the resulting gene list was filtered to identify genes with a 2-fold change in their expression levels (p < 0.05). Gene ontology analysis indicated that the functional categories of TGF-b signaling, lipid metabolism and interferon signaling were affected by ribavirin treatment (p < .009). Quantitative PCR analysis also demonstrated that ribavirin treatment resulted in the induction of a distinct set of ISGs in the HepG2 cell line. When ribavirin was used in conjunction with interferon, induction of specific ISGs was synergistic when compared to either drug applied separately. Although the RIG-I and TLR3 pathways are defective in Huh7.5.1 cells, antiviral signaling was still activated by transfection of polyIC. Several ISGs including IFI44, RSAD2, IFIT1 and 2 were induced by polyIC in both Huh7 and 7.5.1 cells and were also induced following JFH1 infection in Huh7 cells. RNAi mediated suppression of MDA5 inhibited induction of these genes by polyIC and enhanced JFH1 HCV infection. Furthermore, additional augmentation of gene induction was observed with the transfection of polyIC into ribavirin and interferon-treated cells when compared to cells treated with these agents separately. Kinetic analysis demonstrated the greatest gene induction 8 h post-interferon treatment whereas gene induction increased with time after ribavirin and polyIC treatment, further underscoring a difference between ribavirin- and interferon-induced gene induction. In conclusion, our study suggests that ribavirin, acting via a novel innate mechanism, results in the up-regulation of a distinct set of ISGs, which further potentiates the antiviral effects of interferon in the context of HCV infection. doi:10.1016/j.cyto.2009.07.305
PP1-181 The interferon-inducible gene IFI16, a member of the HIN200 family, triggers primary endothelial cell apoptosis through caspase 2 and caspase 3 pathway
Francesca Gugliesi, Marco De Andrea, Michele Mondini, Paola Cappello, Mirella Giovarelli, Marisa Gariglio, Santo Landolfo, Poster Presentation I The interferon-inducible gene IFI16, a member of the HIN200 family, triggers primary endothelial cell apoptosis through caspase 2 and caspase 3 pathway Francesca Gugliesi 1, Marco De Andrea 1,2, Michele Mondini 2,3, Paola Cappello 4, Mirella Giovarelli 4, Marisa Gariglio 2, Santo Landolfo 1, 1 Department of Public Health and Microbiology, University of Turin, Turin, Italy, 2 Department of Clinical and Experimental Medicine, Medical School of Novara, Novara, Italy, 3 NoToPharm SrL, Bioindustry Park del Canavese, Turin, Italy, 4 Department of Medicine and Experimental Oncology, and Center for Experimental Research and Medical Studies, University of Turin, Turin, Italy IFI16, an Interferon (IFN)-inducible gene in humans, is a member of the 200amino acid repeat family of genes HIN200. We previously reported that enforced IFI16 expression in the first 48 h suppressed cell proliferation and resulted in upregulation of inflammatory molecules in human endothelial cells (EC). However, the fate of IFI16-overexpressing EC after inhibition of cell growth has not been clarified so far. Here, we show that IFI16 was sufficient to induce apoptosis, as measured by sub-G1 accumulation, Annexin V binding, and TUNEL analysis in the next 72 h after its overexpression. Activation of caspase 2 and caspase 3 was required for the full apoptotic effect, whereas their inactivation either by specific siRNAs or caspase-2 inhibitor abrogate cell death. IFI16 knockdown by specific siRNA partially prevented EC apoptosis triggered by IFN-beta priming followed by poly rI:rC treatment, proving its physiological relevance in IFN-mediated cell death. Finally, expression of a dominantnegative mutant of IKK2 kinase or treatment with AS602868, an inhibitor of IKK2 activity, halting IkB a degradation and NF-kB activation, resulted in lack of caspase 2 activation and apoptosis induction. We concluded that the IFN-inducible gene IFI16 may contribute to IFN-mediated inhibition of EC cell growth and tube morphogenesis by triggering caspase 2- and caspase 3-mediated programmed cell death through the NF-kB pathway. doi:10.1016/j.cyto.2009.07.304
PP1-182 Characterization of gene induction and antiviral effects on HCVcc following ribavirin, interferon and polyIC stimulation
Emmanuel Thomas, Qisheng Li, Shauna A. Clark, Jordan J. Feld, T. Jake Liang, Poster Presentation I Characterization of gene induction and antiviral effects on HCVcc following ribavirin, interferon and polyIC stimulation Emmanuel Thomas, Qisheng Li, Shauna A. Clark, Jordan J. Feld, T. Jake Liang, NIDDKNIH, Bethesda, MD The combination of interferon-a and ribavirin is the standard treatment for chronic hepatitis C infection. We have previously shown that ribavirin enhances
PP1-184 Polymerization of STAT1 dimers is required for STAT1 nuclear retention and IFNgamma target gene induction
Filipa Antunes, Uwe Vinkemeier, Poster Presentation I Polymerization of STAT1 dimers is required for STAT1 nuclear retention and IFN-c target gene induction Filipa Antunes, Uwe Vinkemeier, School of Biomedical Sciences, The University of Nottingham, Nottingham, UK IFN-c-dependent gene activation requires tyrosine phosphorylation (activation) of STAT proteins mediated by a cell surface receptor. This event leads to their rapid nuclear translocation followed by direct binding to the promoters of responsive genes in the IFN-c activation sites (GAS). Hence, the nuclear import of phosphorylated STAT1 results in their retention in the nucleus. Previous studies have shown that DNA binding controls STAT1 nuclear retention and that disruption of STAT1 polymerization by point mutations compromises IFN-induced gene activation. Here we investigated the role of STAT1 polymerization in nuclear retention, DNA binding and IFN target gene induction. To this effect, STAT1 mutants with different DNA binding and oligomerisation abilities have been generated and characterized by confocal microscopy and time-lapse imaging coupled with photobleaching. Our examination shows that activated STAT1 dimers are not appreciably retained in the nucleus, despite the high affinity DNA binding of the dimers. Accordingly, STAT1 nuclear retention additionally requires the N-domain mediated polymerization of the dimers. To study the physiological role of STAT1 polymerization we generated a polymerization defective knock in mouse model. In our ongoing characterization of the resulted phenotype, we found that for prominent IFN-c target genes, the activated STAT1 is incompetent of generating transcriptional responses, irrespective of the number of confirmed GAS sites. In addition, by using a GFP quantification method, we were able to quantify the number of STAT1 molecules retained in the nucleus due to DNA binding. We conclude that there is a mass deposition of STAT1 polymers on DNA which do not necessarily contains GAS sequences and that STAT1 retention in the nucleus is not limited by the number of DNA binding sites but by the number of activated phosphor dimers. Collectively, our results indicate that phosphor dimers of STAT1 are necessary but not sufficient for IFN-c mediated gene induction. doi:10.1016/j.cyto.2009.07.307
PP1-185 Interferon-induced 20 ,50 -oligoadenylate synthetases versus those from sponges, evolutionarily lowest multicellular animals
Anne Kuusksalu, Annika Lopp, Mailis Päri, Tõnu Reintamm, Merike Kelve, Poster Presentation I Interferon-induced 20 ,50 -oligoadenylate synthetases versus those from sponges, evolutionarily lowest multicellular animals Anne Kuusksalu, Annika Lopp, Mailis Päri, Tõnu Reintamm, Merike Kelve, Department of Gene Technology, Tallinn University of Technology, Tallinn, Estonia
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the induction of interferon-stimulated genes (ISGs) by interferon in vitro and in vivo. Global gene expression profiles generated from the Huh7.5.1 cell line, treated with or without ribavirin, were analyzed using GeneGO and the resulting gene list was filtered to identify genes with a 2-fold change in their expression levels (p < 0.05). Gene ontology analysis indicated that the functional categories of TGF-b signaling, lipid metabolism and interferon signaling were affected by ribavirin treatment (p < .009). Quantitative PCR analysis also demonstrated that ribavirin treatment resulted in the induction of a distinct set of ISGs in the HepG2 cell line. When ribavirin was used in conjunction with interferon, induction of specific ISGs was synergistic when compared to either drug applied separately. Although the RIG-I and TLR3 pathways are defective in Huh7.5.1 cells, antiviral signaling was still activated by transfection of polyIC. Several ISGs including IFI44, RSAD2, IFIT1 and 2 were induced by polyIC in both Huh7 and 7.5.1 cells and were also induced following JFH1 infection in Huh7 cells. RNAi mediated suppression of MDA5 inhibited induction of these genes by polyIC and enhanced JFH1 HCV infection. Furthermore, additional augmentation of gene induction was observed with the transfection of polyIC into ribavirin and interferon-treated cells when compared to cells treated with these agents separately. Kinetic analysis demonstrated the greatest gene induction 8 h post-interferon treatment whereas gene induction increased with time after ribavirin and polyIC treatment, further underscoring a difference between ribavirin- and interferon-induced gene induction. In conclusion, our study suggests that ribavirin, acting via a novel innate mechanism, results in the up-regulation of a distinct set of ISGs, which further potentiates the antiviral effects of interferon in the context of HCV infection. doi:10.1016/j.cyto.2009.07.305
PP1-181 The interferon-inducible gene IFI16, a member of the HIN200 family, triggers primary endothelial cell apoptosis through caspase 2 and caspase 3 pathway
Francesca Gugliesi, Marco De Andrea, Michele Mondini, Paola Cappello, Mirella Giovarelli, Marisa Gariglio, Santo Landolfo, Poster Presentation I The interferon-inducible gene IFI16, a member of the HIN200 family, triggers primary endothelial cell apoptosis through caspase 2 and caspase 3 pathway Francesca Gugliesi 1, Marco De Andrea 1,2, Michele Mondini 2,3, Paola Cappello 4, Mirella Giovarelli 4, Marisa Gariglio 2, Santo Landolfo 1, 1 Department of Public Health and Microbiology, University of Turin, Turin, Italy, 2 Department of Clinical and Experimental Medicine, Medical School of Novara, Novara, Italy, 3 NoToPharm SrL, Bioindustry Park del Canavese, Turin, Italy, 4 Department of Medicine and Experimental Oncology, and Center for Experimental Research and Medical Studies, University of Turin, Turin, Italy IFI16, an Interferon (IFN)-inducible gene in humans, is a member of the 200amino acid repeat family of genes HIN200. We previously reported that enforced IFI16 expression in the first 48 h suppressed cell proliferation and resulted in upregulation of inflammatory molecules in human endothelial cells (EC). However, the fate of IFI16-overexpressing EC after inhibition of cell growth has not been clarified so far. Here, we show that IFI16 was sufficient to induce apoptosis, as measured by sub-G1 accumulation, Annexin V binding, and TUNEL analysis in the next 72 h after its overexpression. Activation of caspase 2 and caspase 3 was required for the full apoptotic effect, whereas their inactivation either by specific siRNAs or caspase-2 inhibitor abrogate cell death. IFI16 knockdown by specific siRNA partially prevented EC apoptosis triggered by IFN-beta priming followed by poly rI:rC treatment, proving its physiological relevance in IFN-mediated cell death. Finally, expression of a dominantnegative mutant of IKK2 kinase or treatment with AS602868, an inhibitor of IKK2 activity, halting IkB a degradation and NF-kB activation, resulted in lack of caspase 2 activation and apoptosis induction. We concluded that the IFN-inducible gene IFI16 may contribute to IFN-mediated inhibition of EC cell growth and tube morphogenesis by triggering caspase 2- and caspase 3-mediated programmed cell death through the NF-kB pathway. doi:10.1016/j.cyto.2009.07.304
PP1-182 Characterization of gene induction and antiviral effects on HCVcc following ribavirin, interferon and polyIC stimulation
Emmanuel Thomas, Qisheng Li, Shauna A. Clark, Jordan J. Feld, T. Jake Liang, Poster Presentation I Characterization of gene induction and antiviral effects on HCVcc following ribavirin, interferon and polyIC stimulation Emmanuel Thomas, Qisheng Li, Shauna A. Clark, Jordan J. Feld, T. Jake Liang, NIDDKNIH, Bethesda, MD The combination of interferon-a and ribavirin is the standard treatment for chronic hepatitis C infection. We have previously shown that ribavirin enhances
PP1-184 Polymerization of STAT1 dimers is required for STAT1 nuclear retention and IFNgamma target gene induction
Filipa Antunes, Uwe Vinkemeier, Poster Presentation I Polymerization of STAT1 dimers is required for STAT1 nuclear retention and IFN-c target gene induction Filipa Antunes, Uwe Vinkemeier, School of Biomedical Sciences, The University of Nottingham, Nottingham, UK IFN-c-dependent gene activation requires tyrosine phosphorylation (activation) of STAT proteins mediated by a cell surface receptor. This event leads to their rapid nuclear translocation followed by direct binding to the promoters of responsive genes in the IFN-c activation sites (GAS). Hence, the nuclear import of phosphorylated STAT1 results in their retention in the nucleus. Previous studies have shown that DNA binding controls STAT1 nuclear retention and that disruption of STAT1 polymerization by point mutations compromises IFN-induced gene activation. Here we investigated the role of STAT1 polymerization in nuclear retention, DNA binding and IFN target gene induction. To this effect, STAT1 mutants with different DNA binding and oligomerisation abilities have been generated and characterized by confocal microscopy and time-lapse imaging coupled with photobleaching. Our examination shows that activated STAT1 dimers are not appreciably retained in the nucleus, despite the high affinity DNA binding of the dimers. Accordingly, STAT1 nuclear retention additionally requires the N-domain mediated polymerization of the dimers. To study the physiological role of STAT1 polymerization we generated a polymerization defective knock in mouse model. In our ongoing characterization of the resulted phenotype, we found that for prominent IFN-c target genes, the activated STAT1 is incompetent of generating transcriptional responses, irrespective of the number of confirmed GAS sites. In addition, by using a GFP quantification method, we were able to quantify the number of STAT1 molecules retained in the nucleus due to DNA binding. We conclude that there is a mass deposition of STAT1 polymers on DNA which do not necessarily contains GAS sequences and that STAT1 retention in the nucleus is not limited by the number of DNA binding sites but by the number of activated phosphor dimers. Collectively, our results indicate that phosphor dimers of STAT1 are necessary but not sufficient for IFN-c mediated gene induction. doi:10.1016/j.cyto.2009.07.307
PP1-185 Interferon-induced 20 ,50 -oligoadenylate synthetases versus those from sponges, evolutionarily lowest multicellular animals
Anne Kuusksalu, Annika Lopp, Mailis Päri, Tõnu Reintamm, Merike Kelve, Poster Presentation I Interferon-induced 20 ,50 -oligoadenylate synthetases versus those from sponges, evolutionarily lowest multicellular animals Anne Kuusksalu, Annika Lopp, Mailis Päri, Tõnu Reintamm, Merike Kelve, Department of Gene Technology, Tallinn University of Technology, Tallinn, Estonia